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ApplicationsofMolecularDiagnosis

forInfectiousDiseases

HouJue

DepartmentofClinicalImmunologyandMicrobiology

INTRODUCTION1.thecommonpathogenofinfectiousdiseases:bacteriafungichlamydiamycoplasmarickettsiaspirochetevirusparasites2.threebasicmethods(1)culturetechnique(conventionalmethod)Morphology:rodorcoccusGramstainpositiveornegative,acidfaststaincolonycharacteristicsBiochemicaltestantibioticsusceptibilitytestMethodologyevaluationhighaccuracy------“goldstandard”IdentifymajoritycommonpathogenLongtimeDifficultorimpossibleculture(2)immunologicaltechniques:

Antibodyandantigendetection

agglutilationprecipitationenzymeimmunoassayImmunofluorescencestaining……MethodologyevaluationhighspecificityandsensitivityRapid,simpleThefalsenegativeresultsThefalsepositiveresults(3)moleculartechnique-newmethod

todetectprimarystructure(sequence)ofDNA(RNA)

HybridizationwithcomplementarysequencesAmplification(synthesis)ofspecificsequences

PCR–polymerasechainreaction

-A-A-T-T-C-G-C-G-A-T-G--T-T-A-A-G-C-G-C-T-A-C-others:RT-PCR,PCR-SSCP,multiplexPCRLCR,NASBAnucleicacidhybridizationgenomeprobegenomegenomegenomeprobeprobeprobeMethodologyevaluationhighsensitivityandspecificityRapidlyDeadoraliveSuitablefor:FewinfectiouscellsSlowgrowingorganismsOrganismswhicharedifficultorcurrentlyimpossibletoculture

ThefalsepositiveandnegativeresultsmayoccurExpensive,needspecialequipmentMolecularDiagnosticsforHIV-11.INTRODUCTIONCurrentestimates>33millionpeoplehaveHIVinfectionApproximately16,000newHIVinfectionsoccurdailyaroundtheworld>700,000peoplehaveHIVinfectioninchinaHIVintheUnitedStatesOver1millionpeoplearelivingwithHIV/AIDSMorethan250,000donotknowtheyareinfectedOnlyhalfofthoseinfectedarereceivingtreatmentApproximately40,000newpeoplegetHIVeachyear2.virologyHIV(humanimmunodeficiencyvirus)HIVisaretrovirus,anRNAvirusTwotypes:HIV-1andHIV-2,TheycancauseAIDSHIV-1isclassifiedintogroupM(major),groupOandgroupNviruses.GroupMcanbedividedintosubtypes(A-K)HIV-2canbedividedintosubtypes(A-G)HIVGroupsandSubtypesSubtypesA-GHIV-2HIVHIV-1GroupOGroupNGroupMSubtypesA-JKHIVgenomeCD4CD4CD4HIVCCR5CCR5chemokineMutantmacrophageCXCR4CD4+T4helpercellsR5orM-tropicX4orT-tropicDual-tropicVirus’stropismThevirusmainlydestroystheCD4+cell,andreducestheirnumbers,ultimatelydevastatetheimmunesystemWhentherearenotenoughCD4cellstoprovideprotectionfromgermsandinfections,HIVinfectioncanprogresstoAIDS(AcquiredImmunoDeficiencySyndrome)AIDSbeginswhentheCD4levelinaperson’sbloodislessthan200cells/mm3thepatientisatriskfromallkindsoflife-threateningopportunisticinfectionsandcancersPneumocystiscarinii(卡氏肺孢子虫)Toxoplasma(弓形虫)Cryptococcous(隐球菌)mycobacteria(分支杆菌)Kaposia(卡氏肉瘤)ThemortalityofAIDSis100%LabDiagnosis:①HIVculturetest

②immunoassaytodetecttheHIVantigen(P24)

oranti-HIV

③moleculartechniquescreeningtestconfirmatoryTestcellculture:HumanTlymphocytes

HIV仅感染表面有CD4受体的细胞。实验室中常用正常人T细胞分离病毒。感染病毒的细胞出现CPEAnimalModels:

primatesAnimals:

恒河猴和黑猩猩solidphasehumansampleconjugateYY1stgen2ndgenHIVEIAassaygenerationsY3rdgenYY4thgenYYYYYYYYYYYY抗原IgG抗体P24抗原YYYYYIgM抗体YELISAforHIVantibodyMicroplateELISAforHIVantibody:colouredwellsindicatereactivityWesternBlot(Immunoblottingtest)HIV-1WesternBlotLane1:PositiveControlLane2:NegativeControlSampleA:NegativeSampleB:IndeterminateSampleC:Positive3.Viralloadtesting

TheamountofHIVinaninfectedpatient’sbloodcalledtheviralload.Theviralloadtestshowshowmuchvirusispresentin thebody.ApersonwithHIVcanhaveaviralloadfromlessthan50copiestoover1,000,000copiespermilliliterofblood(copies/mL)Lessthan50copies/mL=“undetectable”Undetectabledoesnotmeanthepersoniscured

3.1clinicalutilityViralloadtestsareanimportanttoolto:assessdiseaseprogression

Thehigherviralload,themorelikelytoloseCD4cells,themorelikelyprogresstoAIDSinthefuture.determiningwhentoinitiateantiretroviraltherapyCD4Tcellcountsandviralloadlevelsareusedtogethertodeterminewhenyoumayneedtreatment.monitortheresponsetotherapyHIVandAIDSGoodcorrelationbetweennumberofHIVparticlesmeasuredbyPCRandprogressiontodiseasePlasmaHIV-1RNAlevelsaremeasuredimmediatelybeforebeginningtherapy2–8wkafterstartoftherapytodetermineinitialresponse.Testingisrepeatedat3-to4-mointervalstoevaluatecontinuedeffectivenessoftheregimen.Ifthepatienthasadeterioratingclinicalcourse,morefrequenttestingisdonemonitortheresponsetotherapy3.2CommercialtestsAmplicorHIV-1Monitorv1.5Assays(Roche)VersantHIV-1RNA3.0Assay(bDNA)(Bayer)NucliSensHIV-1QTAssay (Organon)FDAapproved

3.2.1BAYERQUANTIPLEXHIV-1RNAV3.0Assay

asignalamplificationtestbDNA(branchDNAassays),hybridizecaptureassaysquantifyplasmaHIV-1byamplifyingthesignalratherthanthetargetRNA

RProcedureTheHIV-1genomicRNAisreleasedfromthevirionsandcapturedontothesurfaceofamicrowellbyasetofimmobilizedHIV-specificoligonucleotidetargetprobes.AsecondsetoftargetprobesareincludedthathybridizetoboththeviralRNAandtothepreamplifierprobes.ThepreamplifierprobeshybridizetothebDNAamplifiers.Multiplecopiesofanalkalinephosphatase-labeledprobecomplementarytotheamplifierDNAsequencearethenhybridizedtotheimmobilizedcomplextofurtheramplifythesignal.Detectionisachievedbyincubatingthecomplexwithachemiluminescentsubstrateandmeasuringthelightemissiongeneratedbytheboundalkalinephosphatase.LightemissionisdirectlyproportionaltotheamountofHIV-1RNApresentineachsample.EachHIV-1RNArunincludesasetofstandardsofknownHIV-1concentration,allowingthedeterminationoftheRNAconcentrationintheclinicalspecimens.VERSANT™HIV-1RNA3.0Assay(bDNA)

bDNA3.0TechnologySchematicRepresentationoftheBranchedDNA(bDNA)AssayforQuantifyingHIV-1(VersantHIV-1RNA3.0)

ThebDNAassaydetectsandquantifiesHIV-1TARGET:polgeneItisFDAapprovedforinvitrodiagnosticuseawidelineardynamicrange(75to500,000copies/mL)ThebDNAassaydoesnotrequireviralRNApurificationorPCRamplificationstepsbDNAishighlyreliableinaccuratelyquantifyingallsubtypesofHIV-13.2.2NucliSensHIV-1RNAQTAssay

TheNucliSensHIV-1QTassay(bioMerieux)isbasedontargetamplificationusingNucleicAcidSequenceBasedAmplification(NASBA)Coamplificationofapatient’sHIV-1RNAsampletogetherwithinternalcalibrators.ThequantityofamplifiedRNAismeasuredbymeansofelectrochemiluminescence(ECL).NucliSensHIV-1QT(NASBA)(electrochemiluminescence,ECL)电化学发光测定HIVQS3QS2QS1亲和素Target:HIV-1gagTheamountofnucleicacidisdetermineddirectlybyelectrochemiluminescenceQuantitationofHIV-1viralloadisplishedusingtheNucliSensReaderbycoamplificationof3internalRNAquantitationstandards(ThreesyntheticRNAsofknowncopynumber,high(Qa104.8),medium(Qb104.2),andlow(Qc103.6),TheyarespikedintotheoriginalspecimenandarecoextractedandcoamplifiedwiththesampleRNAItisFDAapprovedforinvitrodiagnosticusehasabroadlineardynamicrange(176to3,470,000copies/mL),increasingthevolumnofplasmacanimprovedthesensitivity.sensitivity(threshold80copies/ml)Canmodatedifferentspecimenvolumes(0.01~2.0ml,1ml)TheassayisabletoquantifyallHIV-1groupMsubtypesA-Kreliably.(A,C,lower)theentiretestprocedureisinternallycalibratedlowthroughput,modating10-samplebatches3.2.3AmplicorHIV-1Monitorv1.5Assays

aPCR-basedtargetamplificationTarget:gaggeneprocedureViralRNAisreleasedfromthevirionsandextracted.RT-PCRamplificationoccursinasingletubeusingthethermostablebinantenzymeThermusthermophilusDNApolymerase(rTthpol),whichhasbothRTandDNApolymeraseactivities.ThePCRproductsareseriallydilutedanddenatured,andsingle-strandedDNAisboundtomicrowellscoatedwithHIV-specificoligonucleotideprobes.Anavidin-horseradishperoxidase(HRP)conjugateisadded,bindingtothebiotin-labeledamplicon.Theamountofboundampliconisdeterminedusinganenzyme-linkedimmunosorbentassay(ELISA)platereaderaftertheadditionofanHRP-specificcolorimetricsubstrate.ViralRNAisquantifiedusingtheknowninputcopynumberoftheQSRNA杂交/检测(37oC孵育)captureprobes磁珠Avidin-horseradishPeroxidase复和物Biotin扩增子Tetramethylbenzidine(TMB)底物MMPMMPTMBHydrogenPeroxidecoatedwithHIV-specificandQS-specificoligonucleotide

ViralRNAisquantifiedusingasynthetic,noninfectiousRNAastheinternalquantitationstandard(QS),addedtothespecimenataknownconcentrationbeforeRNAextraction.TheinternalQScanbedifferentiatedfromtheviraltargetsequencesandservestocompensateforvariabilityinRNAextractionandtoindicatesubstancesinplasmathatmaybeinhibitorytoPCRamplification.FDAapprovedsensitive,highlyreproducible,andspecificforHIV-1groupMsubtypesA-Htheentiretestprocedureisinternallycalibratedlineardynamicranges:theUltrasensitive(50to100,000copies/mL)theStandard(400to750,000copies/mL)viralloadtestssummaryResultsbetweenthedifferentviral-loadassaysmightnotbecomparable,itisbesttousethesameassaywhenmonitoringpatientsovertime.False-positiveresultscanoccurwithviral-loadassaysandareattributedtolimitationsinassaychemistry,contaminationwithamplicons,orcontaminationduringspecimenprocessing.Eachoftheviral-loadassayshastheirstrengthsandweaknesses.TheVersantbDNAassayhastheadvantagesofhighthroughput,abroaddynamicrange,andtheabilitytoaccuratelyquantifyallsubtypesofHIV-1.However,theassaydoesnothaveaninternalcontroltoevaluatethequalityofnucleicacidextractionandfalse-positiveresultshavebeenreported.TheAmplicorRT-PCRassayhasgoodspecificity,butthedynamicrangeislimited,requiringbothastandardandultra-sensitiveversionoftheassay.TheNASBAassayhasabroaddynamicrangeandcanmodateawiderangeofclinicalspecimentypesandspecimenvolumes,butitunderestimatessomenon-Bsubtypes.

RocheBayer(前Chiron)Organon技术RT-PCRbDNANuclisensHIV-1QT结果比较约2xbDNA2.02.0版结果约为RT-PCR的50%,3.0版结果与Roche基本相同与Roche基本相同优点通过了FDA认证假阳性结果较bDNA少1.5版对A-G亚型有较好的反应性操作时间较少对A-G亚型有较好的反应性通过了FDA认证可使用多种组织,如生殖道分泌液对A-G亚型有较好的反应性检测范围大缺点操作容易污染测定范围较小(相对于NASBA)少数生物样品反应受抑制,如精液、乳汁和唾液早期版本对部分亚型反应性较差,1.0版对非B亚型的样品反应性普遍较差,(5)版通过改进对O亚型检测能力较差对低值样品检测的特异性较差。无内部质量控制,尤其在3.0版中为单孔检测时,无法控制由于提取或加样操作的失误。不同版本间差异较大,2.0版约为RT-PCR50%,3.0版结果与Roche基本相同表明两个版本间差异可能达到50%。对除血浆、血清外的大部分体液样品无法进行检测。对抗凝剂敏感,只能使用EDTA。早期版本(NASBA)对部分亚型(G亚型)反应性差。操作复杂,样品提取(不使用自动提取仪时)和测定反应时需要进行大量的手工操作。不适合一次大量检测,ECL读数仪每次只能进行12个样品的检测。实验内、外误差相对较大。检测范围标准版(Amplicor1.0及1.5)

400-750,000c/mL超敏版(Ultra-Direct1.0and1.5):

50-75,000c/mLbDNA3.0版:100-500,000c/mLNuclisensHIV-1QT:40-10,000,000

c/mL(取决于样品量)各种HIV定量检测方法的比较3.3SpecimenrequirementsPlasmaisthespecimenofchoice.Volume:AmplicorHIV-1Monitor:theUltrasensitive(0.5ml)ortheStandard(0.2ml)VersantHIV-1RNA3.0:1.0mlNucliSensHIV-1Assay:Standard1.0ml,50ul~2.0mlcanbeusedAnticoagulant:EDTAplasmaisthepreferredchoiceofspecimenAmplicor,Versant:EDTANucliSensHIV-1Assay:anyAnticoagulantStore:wholebloodatroomtemperatureshoudbeseparatedplasmafromcellsassoonaspossibleaftercollection(within4-6hours)toavoiddegradationoftheHIVRNA.Store:4°CforseveraldaysForlong-termstorage:Thespecimenshouldbeimmediatelyfrozenatorbelow-70°C(dryice)andtransportedtotheVirologyLaboratoryondryice.4.HIVresistancetestingResistancereferstothereducedsusceptibilityofHIVtoaspecificantiretroviraltherapy(ARV)agenthighlyactiveanti-retroviraltherapy(HAART)NucleosideReverseTranscriptaseInhibitors+Non-nucleosideRTI’s+ProteaseInhibitorsresistantvirusisanimportantcauseoftreatmentfailureChanges(mutations)intheviruscauseresistancemutationsinthereversetranscriptaseorproteasegenes

4.1Resistancetestingutility:canbeusedtoidentifywhichoftheadministereddrugsinaregimenarelikelytobeineffective.*tohelpguidethechoiceofnewanti-HIVregimensfollowingtreatmentfailure,forpregnantwomen,andforpersonswhohavebeeninfectedwithinthepast2yr.

4.2ResistanceTestTypesTherearetwomaintypesofresistancetests:genotypicresistancetestingphenotypicresistancetestingVirtualPhenotype虚拟表型4.3GenotypicResistanceTesting

基因型耐药测试Genotypicassaysdetectmutationsinthereversetranscriptase(RT)orproteasegenes(PR)thatareassociatedwithresistancetooneormoreantiretroviraldrugs.Howthetestisperformed

Step1-Sampleistaken

Step2-Copiesofthevirusgene(UsingPCRtechnology,thegeneticmaterialofaperson'sHIViscopiednumeroustimes)

Step3-Geneswithinthevirusareevaluatedformutations(Onceamplified,particularviralenzymes[mostcommonlythereversetranscriptaseandproteaseenzymes],areevaluatedtodeterminewhethergeneticmutationsarepresent)

Step4-Mutationsfoundinthevirusarecomparedtoknownresistancemutations(Geneticmutationspresentinthepatientsamplearecomparedtopreestablisheddrugresistancemutationpatterns)

Results:Ifthetypeandnumberofmutationspresentinaperson'sHIVmatchpreestablishedmutationpatternsforaparticulardrug,thatindividual'svirushasprobablydevelopedresistancetothatdrug.GenotypicResistanceTesting病人血浆

(>200µl)总RNAcDNA

PR/RT基因(扩增产物)extractionRTPCR

T

CGA

T

GT

A

C

测序序列解释-和野生型病毒序列比较-确定耐药突变位点-解释突变程度及确认耐药种类ViralPR/RTgeneisolationAAAGACAGTAAAAACAGCAAAAACAGCAAAGACAGT密码子无义突变突变基因型检测(Genotypicassays)通过分析HIV反转录酶(RT)基因和蛋白酶(PR)基因的序列来发现耐药性相关点突变LysAspSerLysAsnSer

Genotypictestingmaybeabletodetectthedevelopmentofresistanceearlierthanphenotypictesting,sinceitdetectgeneticmutationsthatmayoccurpriortotheemergenceofphenotypicresistance.Genotypicassaysareavailableinclinicallaboratoriesandaretechnicallyeasiertoperform,haveamorerapidturnaroundtime,andarelessexpensivethanphenotypicassays.However,theinterpretationofgenotypicassaysiscomplex4.4phenotypicresistanceassay表型耐药测试phenotypicresistancetestingmeasureviralreplicationinthepresenceofantiviraldrugs.phenotypingrequiresanHIVviralloadofatleast1,000copies/mL.Phenotypingisalsoabletodetectonlythosemutationsthatcomprise20%ormoreoftheviralpopulation.Currentlyavailablephenotypicassaysrelyonbinanttechnology,phenotypinginvolvesculturingaperson'svirusinthepresenceofdifferentconcentrationsofantiretroviraldrugs.Viralreplicationismeasuredinvaryingconcentrationsofdrug,allowingforthedeterminationoftheamountofdrugrequiredtoinhibitviralreplicationby50%(IC50).TheIC50ofthepatientstrainiscomparedtothatofaWTvirusandfolddifferencesinIC50arereported.inhibit50%ofviralreplication(IC50).Currentlyavailablephenotypicassaysrelyonbinanttechnology,whichallowsmorerapidtestingthantraditionalmethodsthatrequiredisolationofinfectiousvirus.Viralreplicationismeasuredinvaryingcon-centrationsofdrug,allowingforthedeterminationoftheamountofdrugrequiredtoinhibitviralreplicationby50%(IC50).TheIC50ofthepatientstrainiscomparedtothatofaWTvirusandfolddifferencesinIC50arereported.resistanttoantiretroviralscanbedetectedbymeasuringtheamountofdrugnecessarytoinhibitviralgrowthinvitro.ThestandardmeasuresarecalledIC50.ThesedesignationsrefertotheinhibitoryconcentrationofdrugneededtoinhibitthegrowthofHIVby50%.Typically,afour-foldincreaseinIC50,comparedtoalaboratorysampleofwildtype(nonmutated)virus,wouldbetheminimumchangethatcouldbedetected.thecommercialphenotypicresistanceassay:AV(VicroAntivirogram),PS(ViroLogicPhenoSense)

procedurecopythepatient'sHIV

Step1-Asampleofbloodistakenfromthepatient.

Step2-Fromthesample,keyportionsofthegeneswithinthepatient'svirusarecopied.Thisprocessiscalledamplification.

Step3-Oncestep2iscomplete,theamplifiedgenesareinsertedintoalaboratorysampleofHIV.Genessimilartothosecopiedinstep2aremissingfromthelaboratorysample,sothelaboratorysampleofHIVisonlya"shell"thatcannotgrow.

Step4-Whenstep3iscomplete,thelaboratoryHIVesacompletegeneticcopyofthepatient'sHIV.

Results

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